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Note: 12 8-well modules – Each module is designed to break apart for 8 tests.Storage: Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).

Species-Reactivity

Human

Description

The PathScan® Phospho-LAT (Tyr191) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-LAT (Tyr191). A LAT mouse antibody has been coated onto the microwells. After incubation with cell lysates, LAT protein (phosphorylated and non-phosphorylated) is captured by the coated antibody. Following extensive washing, a phospho-LAT (Tyr191) rabbit detection antibody is added to detect the captured phospho-LAT (Tyr191). HRP-linked anti-rabbit antibody is then used to recognize the bound detection antibody. The HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of phospho-LAT (Tyr191).

Antibodies in kit are custom formulations specific to kit

Specificity / Sensitivity

CST's PathScan® Phospho-LAT (Tyr191) Sandwich ELISA Kit detects endogenous levels of Phospho-LAT when phosphorylated at Tyr191. As shown in Figure 1, a significant induction of LAT phosphorylation at Tyr191 can be detected in Jurkat cells following treatment with anti-CD3 using the Phospho-LAT (Tyr191) Sandwich ELISA Kit. The level of total LAT remains unchanged as shown by western analysis (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Sandwich ELISA

Figure 1. Treatment of Jurkat cells with anti-CD3 stimulates phosphorylation of LAT at Tyr191 as detected by the PathScan® Phospho-LAT (Tyr191) Sandwich ELISA Kit #7936, but does not affect the level of total LAT. Jurkat cells were starved for 48 hours and treated with 10 µg/ml anti-CD3 for 2 minutes at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while the western blots, using LAT Antibody #9166 (left panel) or Phospho-LAT (Tyr191) Antibody #3584 (right panel), are shown in the bottom figure.

Sandwich ELISA

Figure 2. The relationship between lysate protein concentration from untreated and anti-CD3-treated Jurkat cells and the absorbance at 450 nm is shown.

Background

LAT, a transmembrane adaptor protein expressed in T, NK and mast cells, is an important mediator for T cell receptor (TCR) signaling (1). Upon TCR engagement, activated Zap-70 phosphorylates LAT at multiple conserved tyrosine residues within SH2 binding motifs, exposing these motifs as the docking sites for downstream signaling targets (2,3). The phosphorylation of LAT at Tyr171 and Tyr191 enables the binding of Grb2, Gads/SLP-76, PLCγ1 and PI3 kinase through their SH2 domain and translocates them to the membrane. This process eventually leads to activation of the corresponding signaling pathways (1-4).